It has previously been reported that gene profiles in surgically-resected colorectal cancer tissues are altered over time possibly due to the different tissue-acquisition methods and sample extraction timing that were used. However, the changes that occur are still not clearly understood. In the present study, time-dependent changes in gene expression profiling in colorectal surgical specimens were analyzed. Normal and tumor tissues at several time-points (0, 30, 60 and 120 min) were extracted, and RNA quality, microarray experiments, quantitative PCR and bioinformatics clustering were performed. Although RNA integrity was preserved 2 h after resection, inherent increased/decreased gene expression was observed from 30-120 min in approximately 10% of genes. Bioinformatics clustering could not distinguish case-by-case, probably due to gene profiling changes. Irregular changes in gene expression after surgical resection were found, which could be a crucial confounding factor for quantitative analyses.